This application requests funds for continuation of support for an already existing Biotechnology Research Resource devoted to EPR spectroscopy. The pulsed EPR instrument was specifically designed for electron spin echo envelope modulation (ESEEM) studies of biological materials. Further, it is unique in that it has wide band capabilities, operating from 8-18 GHz, with proposed core development for operation in the 4-8 GHz range. Other core development of the Resource includes implementation of pulsed ENDOR spectroscopy as an alternative to conventional continuous wave (cw) methods so as to improve sensitivity in the study of magnetically dilute biological materials. A third aspect of core research is the study of single crystal protein samples as a means of defining in greater detail the electron nuclear coupling from individual groups or atoms in a protein structure. This will be applied to the examination of the blue copper proteins, with plastocyanin as an example, where preliminary ENDOR and ESEEM studies demonstrate inequivalence in electron nuclear coupling with the individual copper-coordinated imidazoles that constitute the metal binding site. Other core research will be devoted to the study of Mn(II)-17 0 interactions under exact cancellation conditions where the ESEEM spectrum is dominated by lines arising from nuclear quadrupolar interactions. The results of this study will be applied to the examination of metal substrate interactions with Mn(II)-enzymes. Finally, core research will be devoted to ESEEM studies of electron nuclear coupling to axial and in-plane nitrogen in various lowspin hemoproteins formed with exogenous and endogenous ligands. This project is made possible by the availability of 15N-substituted myoglobin and 15N-heme, so that all axial and equatorial ligands will be studied in the 14N-forms. A number of collaborative studies are proposed to identify and quantify the various metal coordinated ligands in various metalloproteins, the nature of nuclei interacting with free radical centers in biomolecules and the distance to deuterium in specifically labelled substrates and cofactors. LEFE studies are proposed to study the metal composition of iron sulfur clusters. Service and educational activities related to pulsed EPR as a probe of structure of paramagnetic biomolecules are proposed.